Metal-catalyzed oxidation (MCO) of proteins is believed to play an important role in physiopathology and aging. Oxidative damage includes introduction of carbonyl functions to certain amino acid side chains. The present project is aimed at the chemical characterization of protein oxidation products, with special emphasis on protein carbonyls. Since the last progress report, the analytical method for quantitation of adipic semialdehyde was completed. Thus, both glutamic and adipic semialdehydes can now be routinely measured in reduced protein hydrolysates by selected ion monitoring gas chromatography/mass spectrometry after isotopic dilution. Analysis of two model proteins, glutamine synthetase (GS) and bovine serum albumin (BSA), oxidized in vitro indicated that the combined two mentioned semialdehydes make up approximately the totality of protein carbonyls in oxidized GS, while they account for ~60% of protein carbonyls in oxidized BSA. The new procedures, in combination with the classic protein carbonyl assay, proved useful also in studies showing that covalent modifications that enhance the metal binding capacity of proteins make them more susceptible to MCO. Thus, conversion of lysine residues of proteins to carboxymethyl-lysine derivatives, as occurs in patients with diabetes and in aging, led to an increase in MCO-mediated formation of carbonyl groups and adipic/glutamic semialdehyde derivatives. Expanding the studies of rat liver tissue described in the previous report, glutamic and adipic semialdehydes were also measured in human cultured fibroblasts from healthy donors and patients affected by progeria. Also, mitochondrial protein extracts from flies of different ages were analyzed. These studies are providing information on the correlation between concentrations of specific carbonyl products and aging. One important result emerging is that, in contrast to proteins oxidized in vitro, glutamic and adipic semialdehydes represent a much more modest contribution to the total of protein carbonyls. It is also our purpose to study the oxidation of individual proteins. In this respect, studies on the oxidation of recombinant hamster prion protein have been initiated. Preliminary results indicate that MCO induces a dramatic change in the conformation of this protein that aggregates and precipitates. - Metal-catalyzed oxidation, protein carbonyls, aging, glutamic semialdehyde, adipic semialdehyde, carboxymethyl-lysine.